Role of ATM in the telomere response to the G-quadruplex ligand 360A.

Abstract

Telomeres are known to prevent chromosome ends from being recognized as DNA double-strand breaks. Conversely, many DNA damage response proteins, including ATM, are thought to participate to telomere maintenance. However, the precise roles of ATM at telomeres remain unclear due to its multiple functions in cell checkpoints and apoptosis. To gain more insights into the role of ATM in telomere maintenance, we determined the effects of the G-quadruplex ligand 360A in various cell lines lacking functional ATM. We showed, by using Fluorescence in situ hybridization (FISH) and Chromosome Orientation-FISH using telomere PNA probes, that 360A induced specific telomere aberrations occurring during or after replication, mainly consisting in sister telomere fusions and also recombinations that involved preferentially the lagging strand telomeres. We demonstrate that ATM reduced telomere instability independently of apoptosis induction. Our results suggest thus that ATM has a direct role in preventing inappropriate DNA repair at telomeres, which could be related to its possible participation to the formation of protected structures at telomeres.

Cell growth arrest and apoptosis induced by the G-quadruplex ligand 360A in ATM-proficient and deficient immortalized cell lines. (A) Cell growth curves of ATMKD and control HeLa cells, normal (GM03657) and AT (GM03189) EBV-lymphocytes, and, normal (AS3WT2) and AT (GM9607) SV40-fibroblasts treated with 5 µM 360A or 0.05% DMSO as controls. 360A induced cell growth arrest following by massive cell death in all these cell lines. Similar results were obtained at least in three independent experiments. (B) TUNEL labeling of cells treated with 5 µM 360A (or 0.05% DMSO) for 8 days (on the left) or for longer times (11 days or 14 days for SV40-fibroblasts) (on the right). Samples were analyzed by flow cytometry and the proportion of TUNEL-positive cells was expressed as percentage of total population. Data are the means ± SE of at least two independent experiments.

360A induces specific telomere aberrations in HeLa cells that are increased in ATM knocked down cells. Histograms show the percentages of chromosomes with the indicated telomere aberration per cell detected in metaphase spreads of ATMKD and control HeLa cells treated with 5 µM 360A for 8 days (or 0.05% DMSO) and hybridized successively with a telomeric PNA probe (in red) and a centromeric DNA probe (in green) and then counterstained with DAPI (blue). Percentages ± SEs were calculated from n = 30 metaphases per condition. The mean number of chromosomes per cell was 66 and remained unchanged in ATMKD and control HeLa cells. *Indicates a t-test P-value ≤0.05; **P < 0.001; ***P < 0.0001; #indicates a significant difference between DMSO-treated ATMKD and control HeLa cells. Dicentric chromosomes taken into account contained telomere signals at the fused points (No dicentric chromosome without telomere signals at the fused points were detected, see Supplementary Figure 3). Representative images of the different telomere aberrations are presented at the bottom.

The increase of telomere damages concerns all metaphasic cells in 360A-treated ATM-deficient cell cultures. Analysis of the distribution of telomere damages in metaphases from ATMKD and control HeLa cells, normal (GM03657) and AT (GM03189) EBV-lymphocytes, and, normal (AS3WT2) and AT (GM09607) SV40-fibroblasts treated with 5 µM 360A or 0.05% DMSO for 8 days. Box graph shows the distribution of the percentages of damaged telomeres found in each cell (30 to 80 cells per conditions). Boxes include 50% of the values centered on the median (the horizontal line through the box). The vertical lines begin at the 10th percentile and end at the 90th percentile; ***indicates a t-test P-value <0.0001; #indicates a significant difference between ATM-proficient and deficient cells treated with DMSO. Telomere aberrations taken into account were sister chromatid telomere fusions, one telomere losses, telomere doublets, TDM and dicentric chromosomes with telomere signals at fusion points. Terminal deletions were excluded from these analyses since they could also arise from double-strand breaks, although this had no impact on the final results (data not shown).

Respective involvement of lagging and leading telomeres in telomere aberrations induced by 360A revealed by CO-FISH in ATMKD and control HeLa cells. CO-FISH was performed on metaphases of ATMKD and control HeLa cells treated with 5 µM 360A for 8 days (or 0.05% DMSO). Lagging strand telomeres are labeled in red and leading strand telomeres in green. (A) Example of CO-FISH analysis from metaphase spreads of HeLa cells treated with 360A for 8 days showing that sister chromatid fusions involved telomeres of the two chromatids (white arrows and enlarged views) and not telomere-DNA double-strand break fusions. (B) Respective percentages of telomere doublets containing two lagging strand telomeres (G-G doublets) or two leading strand telomeres (C-C doublets) or both lagging and leading strand telomeres (others doublets) in ATMKD and control HeLa cells. n = total number of telomere doublets analyzed. Examples of telomere doublets are shown on the left. Chi-square analysis was performed to detect differences in the repartition of telomere doublets containing two G-G, two C-C or other doublets between the different conditions (**P < 0.001). (C) Percentages of single telomere loss affecting the lagging or the leading strand in ATMKD and control HeLa cells. n = total number of telomere loss analyzed. Examples of missing lagging or leading strand telomeres are shown on the left. Chi-square analysis was performed to detect differences in the repartition of missing lagging or leading strand telomeres between the different conditions (*P < 0.05; **P < 0.001).

360A induces DNA damage signaling. (A–C) 53BP1 and phosphorylated forms (P-) of ATM and SMC1 form foci that co-localized with γ-H2AX following 360A treatment in HeLa cells. HeLa cells were treated with 5 µM 360A (or 0.05% DMSO as control) for 7 days, fixed and costained with anti-γ-H2AX and specific antibodies of 53BP1, P-ATM and P-SMC1. Typical images of 360A-treated HeLa cells exhibited colocalizations between foci of γ-H2AX and DNA repair factors are shown in (A). Histograms (B) show the numbers of P-ATM, γ-H2AX foci and of their colocalization per cell calculated from at least 50 randomly chosen cells for each condition ± SE (***indicate a t-test P-value <0.0001 and **P < 0.005). (C) Colocalizations were quantified by scoring the number of 53BP1 and P-SMC1 foci colocalized or not with γ-H2AX foci in at least 50 randomly chosen cells per condition (***P < 0.0001 and **P < 0.005). (D–F) Colocalization of telomeric-PNA signals (green, in D) and 53BP1 foci (red, in D) in HeLa cells treated with 5 µM 360A (or 0.05% DMSO) for 7 days or exposed to 2 Gy-irradiation (IR) and collected 1 h after irradiation. (D) Colocalization was appreciated on merge images shown on the left for the three conditions. Each image was obtained from a maximum projection of a Z-stack of 15 images, which were previously subjected to 2D deconvolution using the Metamorph software (Universal imaging corp.). Nuclei stained with DAPI (blue) and enlarged views of colocalized foci from the merged image are shown on the right. Percentage of 53BP1 signal colocalized with telomeric PNA signal (E) and percentages of nucleus surfaces occupied by 53BP1 foci (F) in the different conditions were calculated with colocalization module of the Metamorph software (**P < 0.001; ***P < 0.0001).

ATM is required for 360A-induced DNA damage signaling. (A) Percentage of cells with γ-H2AX foci in normal (GM03657) and AT (GM03189) EBV-lymphocyte cell lines treated with 5 µM 360A or 0.05% DMSO for 7 days or exposed to 5-Gy IR as positive controls, then fixed and stained with anti- γ-H2AX antibodies (***P < 0.0001, n > 100). (B, C) Number of γ-H2AX foci per cell in normal (AS3WT2) and AT (GM09607) SV40-fibroblasts (B) and in ATMKD and control HeLa cells (C) treated as described in (A) (**P < 0.001; ***P < 0.0001; n ≥ 50). (D) Number of 53BP1 foci per cell in normal (AS3WT2) and AT (GM09607) SV40-fibroblasts treated as described in (A) (***P < 0.0001, n = 50). (E) Percentage of cells with phospho-SMC1 foci in normal (GM03657) and AT (GM03189) EBV-lymphocytes treated as described in (A) and immunostained with anti-phospho-SMC1 antibodies (***P = 0.0002, n > 100).

Model for 360A-induced telomere aberrations and the role of ATM in telomere stability. (A) 360A destabilizes telomere T-loops in G1 or early S by inducing or stabilizing G-quadruplexes at G-overhangs leading either to overhang degradation, or to inappropriate strand-invasion into interstitial telomere-related sequences in cis forming a large T-loop. In the first case, blunt-ended telomeres of distinct chromosomes can be fused by NHEJ, forming characteristic dicentric chromosomes at metaphase containing parental telomere strands at the fusion points (in yellow) (). In the second case, which could be favored by 360A-induced G-quaduplexes at interstitial sites, recombination between telomere and interstitial sequences generates at metaphase a terminally deleted chromosome and a TDM corresponding to the large T-loop (). (B) G-quadruplex formation at parental G-overhang prevents formation of a stable and protected telomere structure in late S and G2. This could lead either to overhang degradation, unstable T-loop formation or inappropriate overhang-invasion into interstitial sequences in cis. In the first case, blunt-ended telomere of the lagging strand can be fused by NHEJ with sister chromatid before generation of the leading strand G-overhang (alternatively leading strand telomere G-overhang could be degraded as a consequence of G-quadruplex formation) producing a metaphase chromosome with telomere sister fusion (in yellow). In the second case, unstable T-loop could undergo T-loop HR (), which would result at metaphase in a chromosome lacking its lagging strand telomere and an extra chromosome signal containing parental telomere G-strands (in red). In the third case, recombination between lagging strand chromosomes and interstitial sequences in cis produces telomere doublets made of two signals of the parental G strand (in red) separated by interstitial DNA sequences, although the precise mechanism involved is unknown. ATM could attenuate telomere instability by insuring the reformation of stable and protected structures at destabilized telomeres and after replication through its participation in the process of generation of T-loop structures. Parental G strands are in red, parental C strands in green and neosynthesized strands in black. Centromeres are represented as black ellipses.